WO2025177220A1

WO2025177220A1 - Alternative self-amplifying rna

Info

Publication number: WO2025177220A1
Application number: PCT/IB2025/051865
Authority: WO
Inventors: Cheng Chang; Asha PHILIP
Original assignee: Seqirus Inc
Current assignee: Seqirus Inc
Priority date: 2024-02-21
Filing date: 2025-02-21
Publication date: 2025-08-28
Anticipated expiration: 2026-08-21

Abstract

The present disclosure provides a self-amplifying RNA (sa-mRNA) comprising an open reading frame encoding a replicase or a non-structural protein 4 (nsP4) and an open reading frame encoding peptide or polypeptide, wherein the open reading frame encoding the peptide or polypeptide is co-expressed with the replicase or nsP4.

Description

ALTERNATIVE SELF-AMPLIFYING RNA

RELATED APPLICATION DATA

The present application claims priority from United States Patent Application No. 63/556,098 filed 21 February 2024 entitled “Alternative self-amplifying RNA”. The entire contents of this application are hereby incorporated by reference.

SEQUENCE LISTING

The present application is filed together with a Sequence Listing in electronic form. The entire contents of the Sequence Listing are hereby incorporated by reference.

FIELD

The present disclosure relates to an alternative self-amplifying RNA platform with high transfectability.

BACKGROUND

Respiratory viral infections are a significant threat to human health and lives. Infections, such as those caused by the influenza virus and severe acute respiratory syndrome coronavirus (SARS-CoV) have been known to cause global pandemics, killing millions of people worldwide.

Currently, infections such as influenza are treated with antivirals or other drugs. However, there are currently no specific and effective treatments for most respiratory viral infections. For those specific treatments available for some respiratory viral infection, for example, mRNA vaccines against COVID- 19, further improvements can be made to increase their efficacy.

Traditional self-amplifying mRNA (sa-mRNA) platform for gene delivery or vaccine application depends on the production of amplified subgenomic transcript from the negative strand to enable recombinant gene expression. The traditional sa-mRNA’ s kinetic is following: Firstly, genomic RNA replicates into a negative strand genomic RNA, then negative strand genomic RNA serves as a template for amplified subgenomic transcript production and for genomic replication. Lastly, subgenomic transcript produces protein of interest. Although this process allows for reliable expression of the subgenomic transcript, it requires multiple steps for the transcript to be expressed.

Thus, there remains a need for the development of a more efficient sa-mRNA platform, for gene delivery or vaccine application, e.g., for the treatment or prevention of viral infections. Therefore, it will be apparent to the skilled person that there is a need in the art for a sa-mRNA vaccine with improved transfectability and efficient expression of a gene of interest.

SUMMARY

The inventors of the present disclosure have produced a self-amplifying messenger RNA (sa-mRNA) that directly links the gene of interest with the genomic replicon, without the need for a subgenomic promoter. This permits expression of the gene of interest along with the genomic replicon and removes the extra step of amplifying the subgenomic transcript.

The findings by the inventors provide the basis for an alternative self-amplifying RNA, e.g., for therapy, e.g., a self- amplifying RNA vaccine. The findings by the inventors also provide the basis for a mono-cistronic or multi-cistronic self-amplifying RNA, e.g., a mono-cistronic or multi-cistronic self-amplifying RNA vaccine. For example, an open reading frame encoding a protein of interest can be linked to expression of the genomic replicon, e.g., replicase and an open reading frame encoding another protein of interest can be linked to a subgenomic promoter Furthermore, the findings by the inventors provide the basis for methods of treating a disease or condition (e.g., a disease caused by a respiratory viral infection, such as influenza, a SARS-COV-2 infection, COVID-19 or ARDS, RSV, influenza, PIV3, hMPV or EBV) in a subject. The findings also provide the basis for expressing a therapeutic protein and/or a gene editing protein in a subject.

The present disclosure provides a sa-mRNA comprising an open reading frame encoding a replicase or a non- structural protein 4 (nsP4) and an open reading frame encoding peptide or polypeptide, wherein the open reading frame encoding the peptide or polypeptide is co-expressed with the replicase or nsP4.

In one example, protein or peptide is expressed as a fusion protein with the replicase or the nsP4 or the open reading frame encoding the protein or peptide is linked with an internal ribosome entry site (IRES).

For example, the open reading frame encoding the peptide or polypeptide is linked with an internal ribosome entry site (IRES) that induces co-expression with the replicase or nsP4.

Accordingly, the present disclosure provides a polynucleotide comprising an open reading frame encoding a replicase or a non- structural protein 4 (nsP4) and an open reading frame encoding peptide or polypeptide, wherein the sequence encoding the peptide or polypeptide is linked with an internal ribosome entry site (IRES) that induces co-expression with the replicase or nsP4. For example, the open reading frame encoding the replicase or nsP4 is linked with the open reading frame encoding peptide or polypeptide with an IRES.

In one example, the IRES is selected from the group consisting of an EMCV IRES, a FGF1 IRES, a FGF2 IRES, a PDGF IRES, a VEGF IRES or an IGF2 IRES. For example, the IRES is an EMCV IRES. For example, the IRES is a FGF1 IRES. For example, the IRES is a FGF2 IRES. For example, the IRES is a PDGF IRES. For example, the IRES is a VEGF IRES. For example, the IRES is an IGF2 IRES.

In one example, the IRES is an EMCV 4A IRES, an EMCV 5A IRES, an EMCV 6A IRES, an EMCV 7A IRES, an EMCV 8A IRES, an EMCV 10A IRES or an EMCV 12A IRES. For example, the IRES is an EMCV 4A IRES. For example, the IRES is an EMCV 5 A IRES. For example, the IRES is an EMCV 6A IRES. For example, the IRES is an EMCV 7A IRES. For example, the IRES is an EMCV 8 A IRES. For example, the IRES is an EMCV 10A IRES. For example, the IRES is an EMCV 12A IRES.

In one example, EMCV 6A IRES comprises a sequence as set forth in SEQ ID NO: 1.

In one example, the protein or peptide is expressed as a fusion protein with the replicase or the nsP4 and the fusion protein comprises a protease cleavage site positioned between the protein or peptide and the replicase or the nsP4.

In one example, the protein or peptide is expressed as a fusion protein with the replicase or the nsP4 and the fusion protein comprises a self-cleaving peptide positioned between the protein or peptide and the replicase or the nsP4.

In one example, the self-cleaving peptide is a 2A peptide.

In one example, the self-cleaving peptide is T2A, P2A, E2A or F2A. For example, the self-cleaving peptide is T2A. For example, the self-cleaving peptide is P2A For example, the self-cleaving peptide is E2A. For example, the self-cleaving peptide is F2A.

In one example, the self-cleaving peptide is T2A, comprising a sequence as set forth in SEQ ID NO: 5.

In one example, the polynucleotide comprises in 5’ to 3’ order: an open reading frame encoding a replicase or a nsP4, an IRES, an open reading frame encoding a peptide or polypeptide, wherein the peptide or polypeptide is co-expressed with the replicase or nsP4.

In one example, the polynucleotide comprises in 5’ to 3’ order: an open reading frame encoding a replicase or a nsP4, an EMCV IRES, an open reading frame encoding a peptide or polypeptide, wherein the peptide or polypeptide is co-expressed with the replicase or nsP4. In one example, the polynucleotide comprises in 5’ to 3’ order: an open reading frame encoding a replicase or a non-structural protein 4 (nsP4), a sequence encoding a self-cleaving peptide, an open reading frame encoding a peptide or polypeptide.

In one example, the polynucleotide comprises in 5’ to 3’ order: an open reading frame encoding a replicase or a non-structural protein 4 (nsP4), a sequence encoding a T2A self-cleaving peptide, an open reading frame encoding a peptide or polypeptide.

In one example, the polynucleotide is multicistronic. For example, the polynucleotide comprises an open reading frame encoding a replicase or nsP4 and two or more open reading frames encoding peptides or polypeptides, wherein the sequences encoding the peptides or polypeptides are linked with internal ribosome entry sites (IRES) that induces co-expression with the replicase or nsP4.

For example, the multicistronic polynucleotide comprises sequences encoding two or three or four peptides or polypeptides.

In one example, the multicistronic polynucleotide comprises in 5’ to 3’ order: an open reading frame encoding a replicase or a nsP4, an IRES, an open reading frame encoding a first peptide or polypeptide and an open reading frame encoding a second peptide or polypeptide, wherein the first and second peptides or polypeptides are coexpressed with the replicase or nsP4.

In one example, the polynucleotide comprises in 5’ to 3’ order: an open reading frame encoding a replicase or a nsP4, a first IRES, a first open reading frame encoding a peptide or polypeptide, a second IRES, a second open reading frame encoding a peptide or polypeptide wherein the first and second peptides or polypeptides are co-expressed with the replicase or nsP4.

The first and second IRES can be the same or different.

In one example, the polynucleotide comprises in 5’ to 3’ order: an open reading frame encoding a replicase or a nsP4, an EMCV IRES, a first open reading frame encoding a peptide or polypeptide, an EMCV IRES, a second open reading frame encoding a peptide or polypeptide, wherein the first and second peptides or polypeptides are co-expressed with the replicase or nsP4.

In one example, the multicistronic polynucleotide comprises in 5’ to 3’ order: an open reading frame encoding a replicase or a nsP4, a sequence encoding a self-cleaving peptide, an open reading frame encoding a first peptide or polypeptide and an open reading frame encoding a second peptide or polypeptide, wherein the first and second peptides or polypeptides are co-expressed with the replicase or nsP4.

In one example, the polynucleotide comprises in 5’ to 3’ order: an open reading frame encoding a replicase or a nsP4, a sequence encoding a first self-cleaving peptide, a first open reading frame encoding a peptide or polypeptide, a sequence encoding a second self-cleaving peptide, a second open reading frame encoding a peptide or polypeptide wherein the first and second peptides or polypeptides are co-expressed with the replicase or nsP4.

In one example, the multi ci str onic polynucleotide comprises in 5’ to 3’ order: an open reading frame encoding a replicase or a nsP4, a sequence encoding an IRES, an open reading frame encoding a first peptide or polypeptide and an open reading frame encoding a second peptide or polypeptide, wherein the first and second peptides or polypeptides are co-expressed with the replicase or nsP4.

In one example, the polynucleotide comprises in 5’ to 3’ order: an open reading frame encoding a replicase or a nsP4, an IRES, a first open reading frame encoding a peptide or polypeptide, a sequence encoding a self-cleaving peptide, a second open reading frame encoding a peptide or polypeptide wherein the first and second peptides or polypeptides are co-expressed with the replicase or nsP4.

In one example, the polynucleotide comprises in 5’ to 3’ order: an open reading frame encoding a replicase or a nsP4, a sequence encoding a self-cleaving peptide, a first open reading frame encoding a peptide or polypeptide, an IRES, a second open reading frame encoding a peptide or polypeptide wherein the first and second peptides or polypeptides are co-expressed with the replicase or nsP4.

In one example, the multicistronic polynucleotide comprises in 5’ to 3’ order: an open reading frame encoding a replicase or a nsP4, an IRES, an open reading frame encoding a first peptide or polypeptide a SGP and an open reading frame encoding a second peptide or polypeptide, wherein the first peptide or polypeptide is co-expressed with the replicase or nsP4.

In one example, the multicistronic polynucleotide comprises in 5’ to 3’ order: an open reading frame encoding a replicase or a nsP4, a sequence encoding a self-cleaving peptide, an open reading frame encoding a first peptide or polypeptide a SGP and an open reading frame encoding a second peptide or polypeptide, wherein the first peptide or polypeptide is co-expressed with the replicase or nsP4.

In one example, the peptide or protein is a therapeutic protein or peptide is selected from the group consisting of

(i) therapeutic proteins for use in the treatment of cancer or tumor diseases;

(ii) therapeutic proteins for use in enzyme replacement therapy for the treatment of metabolic, endocrine or amino acid disorders or for use in replacing an absent, deficient or mutated protein; (iii) therapeutic proteins for use in the treatment of blood disorders, diseases of the circulatory system, diseases of the respiratory system, infectious diseases or immune deficiencies;

(iv) therapeutic proteins for use in hormone replacement therapy;

(v) therapeutic proteins for use in reprogramming somatic cells into pluri- or omnipotent stem cells;

(vi) therapeutic proteins for use as adjuvant or immuno stimulation;

(vii) therapeutic proteins being a therapeutic antibody;

(viii) therapeutic proteins being a gene editing agent; or

(ix) an antagonist of adaptive immunity, e.g., an interferon antagonist.

In one example, the peptide or polypeptide are antigen(s) from an infectious organism.

In one example, the infectious organism is a virus.

In one example, the virus causes a respiratory condition.

In one example, the virus is a coronavirus or respiratory syncytial virus (RSV) or human metapneumo virus (hMPV) or parainfluenza virus (PIV). For example, the virus is a coronavirus. For example, the virus is a respiratory syncytial virus (RSV). For example, the virus is a human metapneumo virus (hMPV). For example, the virus is a parainfluenza virus (PIV).

In one example, the antigen is selected from:

(a) Spike protein from a coronavirus or a prefusion stabilized form thereof or a receptor binding domain thereof;

(b) F protein from RSV or a prefusion stabilized form thereof;

(c) F protein from hMPV or a prefusion stabilized form thereof;

(d) F protein from PIV3 or a prefusion stabilized form thereof;

(e) Hemagglutinin from influenza; or

(f) Neuraminidase from influenza.

For example, the antigen is Spike protein from a coronavirus or a prefusion stabilized form thereof or a receptor binding domain thereof. For example, the antigen is F protein from RSV or a prefusion stabilized form thereof (e.g., DS-Cavl). For example, the antigen is F protein from hMPV or a prefusion stabilized form thereof. For example, the antigen is F protein from PIV3 or a prefusion stabilized form thereof. For example, the antigen is Hemagglutinin from influenza. For example, the antigen is Neuraminidase from influenza. In on example, the polynucleotide is multicistronic and comprises an open reading frame encoding hemagglutinin from influenza and an open reading frame encoding a neuraminidase from influenza.

In on example, the polynucleotide additionally comprises one or more of the following:

(a) an open reading frame encoding spike protein from a coronavirus or a prefusion stabilized form thereof or a receptor binding domain thereof;

(b) an open reading frame encoding F protein from RSV or a prefusion stabilized form thereof;

(c) an open reading frame encoding F protein from hMPV or a prefusion stabilized form thereof; or

(d) an open reading frame encoding F protein from PIV3 or a prefusion stabilized form thereof.

For example, the polynucleotide additionally comprises one or more of an open reading frame encoding a spike protein from a coronavirus. For example, the polynucleotide additionally comprises one or more of an open reading frame encoding F protein from RSV. For example, the polynucleotide additionally comprises one or more of an open reading frame encoding F protein from hMPV. For example, the polynucleotide additionally comprises one or more of an open reading frame encoding F protein from PIV3.

Reference to any specific protein herein also encompasses a soluble form thereof, e.g., lacking a cytoplasmic and/or intracellular domain.

In one example, the polynucleotide is RNA. For example, the polynucleotide is a sa-mRNA.

The present disclosure provides a method of expressing a peptide or polypeptide in a subject, the method comprising administering the sa-mRNA, wherein the peptide or polypeptide is encoded by the open reading frame(s) linked the replicase or nsP4.

The present disclosure provides a method of treating a disease or condition, the method comprising administering the sa-mRNA, wherein a therapeutic peptide or polypeptide is encoded by the open reading frame(s) linked to the replicase or nsP4 and expression of the therapeutic peptide or polypeptide treats the disease or condition.

The present disclosure provides a method of inducing an immune response in a subject, the method comprising administering the sa-mRNA, wherein an antigen is encoded by the open reading frame(s) linked to the replicase or nsP4 and expression of the antigen induces the immune response. The present disclosure provides a use of the sa-mRNA in the manufacture of a medicament for expressing a peptide or polypeptide in a subject.

The present disclosure provides a use of the sa-mRNA in the manufacture of a medicament for treating a disease or condition in a subject.

The present disclosure provides a use of the sa-mRNA in the manufacture of a medicament for inducing an immune response in a subject.

The present disclosure provides the sa-mRNA for use use in expressing a peptide or polypeptide in a subject.

The present disclosure provides the sa-mRNA for use in treating a disease or condition in a subject.

The present disclosure provides the sa-mRNA for use in inducing an immune response in a subject.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. Representation of the vectors. A) F500.3 control vector (Replicase- SGP-H5). B) F903 SGP deletion vector (Replicase-IRES-H5).

Figure 2. Shows H5 RNA activity in BHK-21 cells as measured by FACS. A) H5 RNA activity. B) Geometric mean fluorescence intensity of H5 RNA.

Figure 3. Shows H5 RNA activity in C2C12 cells as measured by FACS (Experiment 1). A) H5 RNA activity. B) Geometric mean fluorescence intensity of H5 RNA.

Figure 4. Shows H5 RNA activity in HEK-293 cells as measured by FACS (Experiment 2). A) H5 RNA activity. B) Geometric mean fluorescence intensity of H5 RNA.

Figure 5. Shows a summary of H5 RNA activity assays as measured by FACS.

Figure 6. Representation of different sa-mRNA constructs.

Figure 7. A) Representation of sa-mRNA constructs. B) Gel of linearized constructs. C) Gel of IVT RNA expression. Figure 8. Shows NS1 co-expression enhances H5 activity in Hela cells. A) Natural log fo vs ng sa-mRNA (%H5+). B) GMFI-APC.

Figure 9. Shows NS1 co-expression enhances H5 activity in MRC5 cells. A) Activity assay in MCR5. B) GMFI APC (HA).

Figure 10. Shows H5 RNA activity in BHK21 cells. A) Natural log fo vs ng sa- mRNA (%H5+). B) GMFI.

Figure 11. Shows NS1 expression in MRC5 cells.

Figure 12. Shows RNA activity in immunocompetent cells (MRC5 and C2C12).

Figure 13. Representation of sa-mRNA constructs with different antigen in the presence of NS1.

Figure 14. Representation of sa-mRNA constructs with T-cell antigens in the presence of NS1.

Figure 15. Shows H5 RNA activity in C2C12 cells (trial 1). A) Natural log fo vs ng sa-mRNA (%H5+). B) GMFI-APC.

Figure 16. Shows H5 RNA activity in C2C12 cells (trial 2 part 1). A) Natural log fo vs ng sa-mRNA (%H5+). B) GMFI-APC.

Figure 17. Shows H5 RNA activity in C2C12 cells (trial 2 part 2). A) Natural log fo vs ng sa-mRNA (%H5+). B) GMFI-APC.

Figure 18. Shows H5 RNA activity in MRC5 cells (trial 1). A) Natural log fo vs ng sa-mRNA (%H5+). B) GMFI-APC.

Figure 19. Shows H5 RNA activity in MRC5 cells (trial 1). A) Natural log fo vs ng sa-mRNA (%H5+). B) GMFI-APC.

Figure 20. Shows H5 RNA activity in MRC5 cells (trial 2 batch 1). A) Natural log fo vs ng sa-mRNA (%H5+). B) GMFI-APC. Figure 21. Shows H5 RNA activity in MRC5 cells (trial 2 batch 2). A) Natural log fo vs ng sa-mRNA (%H5+). B) GMFI-APC.

Figure 22. Shows a comparison of H5 RNA activity in C2C12 and MRC5 cells.

KEY TO SEQUENCE LISTING

SEQ ID NO: 1 Nucleotide sequence of EMCV 6A IRES

SEQ ID NO: 2 Nucleotide sequence of nsp4/replicase

SEQ ID NO: 3 Amino acid sequence of nsp4/replicase

SEQ ID NO: 4 Nucleotide sequence of F903

SEQ ID NO: 5 Amino acid sequence of T2A self-cleaving peptide

The skilled person will appreciate that a sequence herein that is a RNA will have U in place of T and such sequences are contemplated by the present disclosure as are modified forms of nucleotides, such as pseudouridine or Nl-methyl-pseudouridine.

DETAILED DESCRIPTION

General

Throughout this specification, unless specifically stated otherwise or the context requires otherwise, reference to a single step, composition of matter, group of steps or group of compositions of matter shall be taken to encompass one and a plurality (i.e. one or more) of those steps, compositions of matter, groups of steps or groups of compositions of matter.

Those skilled in the art will appreciate that the present disclosure is susceptible to variations and modifications other than those specifically described. It is to be understood that the disclosure includes all such variations and modifications. The disclosure also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations or any two or more of said steps or features.

The present disclosure is not to be limited in scope by the specific examples described herein, which are intended for the purpose of exemplification only. Functionally-equivalent products, compositions and methods are clearly within the scope of the present disclosure.

Any example of the present disclosure herein shall be taken to apply mutatis mutandis to any other example of the disclosure unless specifically stated otherwise. Stated another way, any specific example of the present disclosure may be combined 10 with any other specific example of the disclosure (except where mutually exclusive).

Any example of the present disclosure disclosing a specific feature or group of features or method or method steps will be taken to provide explicit support for disclaiming the specific feature or group of features or method or method steps.

Unless specifically defined otherwise, all technical and scientific terms used herein shall be taken to have the same meaning as commonly understood by one of ordinary skill in the art (for example, in cell culture, molecular genetics, immunology, immunohistochemistry, protein chemistry, and biochemistry).

Unless otherwise indicated, the recombinant protein, cell culture, and immunological techniques utilized in the present disclosure are standard procedures, well known to those skilled in the art. Such techniques are described and explained throughout the literature in sources such as, J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et al. Molecular Cloning: A Laboratory Manual, Cold Spring Harbour Laboratory Press (1989), T.A. Brown (editor), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991), D.M. Glover and 25 B.D. Hames (editors), DNA Cloning: A Practical Approach, Volumes 1-4, IRL Press (1995 and 1996), and F.M. Ausubel et al. (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all updates until present), Ed Harlow and David Lane (editors) Antibodies: A Laboratory Manual, Cold Spring Harbour Laboratory, (1988), and J.E. Coligan et al. (editors) Current Protocols in Immunology, John Wiley & Sons (including all updates until present).

The term “and/or”, e.g., “X and/or Y” shall be understood to mean either “X and Y” or “X or Y” and shall be taken to provide explicit support for both meanings or for either meaning.

Throughout this specification the word “comprise”, or variations such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

As used herein the term “derived from” shall be taken to indicate that a specified integer may be obtained from a particular source albeit not necessarily directly from that source. Similarly, the term “based on” shall be taken to indicate that a specified integer may be developed or used from a particular source albeit not necessarily directly from that source.

All publications cited herein are hereby incorporated by reference in their entirety. Where reference is made to a URL or other such identifier or address, it is understood that such identifiers can change and particular information on the internet can come and go, but equivalent information can be found by searching the internet. Reference thereto evidences the availability and public dissemination of such information.

Any discussion of documents, acts, materials, devices, articles or the like that has been included in the present specification is solely for the purpose of providing a context for the present invention. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed before the priority date of each claim of this application.

Selected Definitions

As used herein, the terms “self-amplifying mRNA”, “sa-mRNA” or “selfreplicating RNA” refer to a construct based on an RNA virus that has been engineered to allow expression of heterologous RNA and proteins. Self-amplifying mRNA (e.g., in the form of naked RNA) can amplify in host cells leading to expression of the desired gene product in the host cell.

As used herein, the term “polynucleotide” or “nucleotide sequence” or “nucleic acid sequence” will be understood to mean a series of contiguous nucleotides (or bases) covalently linked to a phosphodiester backbone. By convention, sequences are presented from the 5' end to the 3' end, unless otherwise specified.

As used herein, the term “peptide”, “polypeptide” or “polypeptide chain” will be understood to mean a series of contiguous amino acids linked by peptide bonds. For example, a protein shall be taken to include a single polypeptide chain i.e., a series of contiguous amino acids linked by peptide bonds or a series of polypeptide chains covalently or non-covalently linked to one another (i.e., a polypeptide complex). The series of polypeptide chains can be covalently linked using a suitable chemical or a disulfide bond. Examples of non-covalent bonds include hydrogen bonds, ionic bonds, Van der Waals forces, and hydrophobic interactions.

As used herein, the term “subgenomic promoter” (SG promoter; also known as ‘junction region’ promoter) refers to a promoter that directs the expression of a heterologous nucleotide sequence, regulating protein expression.

As used herein, the term “linked to” means positioning a subgenomic promoter or regulatory element (e.g., an IRES) relative to a nucleic acid such that expression of the nucleic acid is controlled or regulated by the element. For example, a subgenomic promoter can be operably linked to numerous nucleic acids, e.g., through another regulatory element, such as an internal ribosome entry site (IRES). As used herein, the term “multi-cistronic” (also known as “polycistronic”) in reference to the polynucleotide, RNA, cRNA and/or self-replicating or amplifying RNA, refers to a RNA that encodes two or more polypeptides. The term encompasses “bicistronic” (or “dicistronic”; i.e., encoding two polypeptides), “tricistronic” (i.e., encoding three polypeptides) and “quadci str onic” (i.e. encoding four polypeptides) molecules. By “bicistronic” is meant a single nucleic acid that is capable of encoding two distinct polypeptides from different regions of the nucleic acid.

As used herein, the term “antigen” refers to a molecule or structure containing one or more epitopes that induce, elicit, augment or boost a cellular and/or humoral immune response. Antigens can include, for example, proteins and peptides from a pathogen such as a virus, bacteria, fungus, protozoan, plant or from a tumour.

As used herein, the terms “treating”, “treat” or “treatment” include administering a RNA or composition described herein to thereby reduce or eliminate at least one symptom of a specified disease or condition.

As used herein, the term “preventing”, “prevent” or “prevention” includes providing prophylaxis with respect to occurrence or recurrence of a specified disease or condition in an individual. An individual may be predisposed to or at risk of developing the disease but has not yet been diagnosed with the disease.

As used herein, the term “subject” shall be taken to mean any animal including humans, for example a mammal. Exemplary subjects include but are not limited to humans and non-human primates. For example, the subject is a human, which can be an infant, a child, an adult or an elderly adult.

As used herein, the terms “disease”, “disorder” or “condition” refers to a disruption of or interference with normal function, and is not to be limited to any specific condition, and will include diseases or disorders.

Self-amplifying mRNA

The present disclosure provides a sa-mRNA (also known as a replicon). For example, the present disclosure provides a multicistronic sa-mRNA.

The skilled person will understand that the sa-mRNA of the present disclosure is based on the genomic RNA of RNA viruses. The RNA should be positive (+)-stranded so that it can be directly translated after delivery to a cell without the need for intervening replication steps (e.g., reverse transcription). Translation of the RNA results in the production of non- structural proteins (NSPs) which combine to form a replicase complex (i.e., an RNA-dependent RNA polymerase). The peptide or polypeptide of interest is expressed with the replicase complex by virtue of the encoding sequence being linked to an IRES that directed expression with the replicase. The complex then amplifies the original RNA, producing both antisense and sense transcripts, resulting in production of multiple daughter RNAs which may subsequently be translated and transcribed, enhancing overall protein expression.

In one example, the sa-mRNA of the present disclosure comprises an open reading frame encoding the non- structural proteins of the RNA virus linked to an IRES which is in turn linked to a sequence encoding a peptide and a polypeptide, and a 5’ and 3’ untranslated regions (UTRs).

In one example, the sa-mRNA comprises an open reading frame encoding one or more non- structural proteins of the RNA virus. For example, the RNA comprises at least one or more genes selected from the group consisting of a viral replicase (or viral polymerase), a viral protease, a viral helicase and other non-structural viral proteins. For example, the sa-mRNA comprises a viral replicase (or viral polymerase).

It will be apparent to the skilled person that RNA suitable for use in the present disclosure may also include a 5' untranslated region (5’-UTR), a 3' untranslated region (3’UTR), and/or a coding or translating sequence. In addition, the RNA may comprise a 5' cap structure, a chain terminating nucleotide, a stem loop (e.g., a histone stem loop), a 3’ tailing sequence (e.g., a polyadenylation signal or one or more polyA tails). Typically, the sa-mRNA of the disclosure comprises, in order from 5’ to 3’ : a 5’cap structure, a 5’-UTR, nucleotide sequences encoding non-structural proteins (NSPs), an IRES, a nucleotide sequence encoding a polypeptide of interest, a 3’-UTR, a fragment and/or a variant thereof and a tailing sequence (e.g. a polyadenylation signal or poly-A tail).

Subgenomic Promoter

The present disclosure provides a self-amplifying mRNA comprising an open reading frame encoding an antigen operably linked to a SG promoter.

SG promoters (also known as ‘junction region’ promoters) suitable for use in the present disclosure will be apparent to the skilled person and/or are described herein.

In one example, the SG promoter is derived from or based on an alphavirus SG promoter. For example, the SG promoter is a native alphavirus SG promoter. In one example, the native SG promoter is a minimal SG promoter. For example, the minimal SG promoter is the minimal sequence required for initiation of transcription. In one example, the native SG promoter is an extended SG promoter. For example, the extended SG promoter is a minimal SG promoter extended at the 5’ end with nucleotides occurring in a sequence encoding a non-structural protein (e.g., nsp4) of the RNA virus (e.g., an alphavirus). In one example, the extended SG promoter is a minimal SG promoter extended at the 5’ end with nucleotides occurring in a sequence encoding an alphavirus NSP4.

In one example, the polynucleotide of the disclosure comprises a SG promoter from any alphavirus. For example, the sa-mRNA of the disclosure comprises a SG promoter from any alphavirus.

In one example, the self-replicating RNA comprises a SG promoter from any alphavirus.

The sa-mRNA of the present disclosure comprises two or more nucleotide sequences encoding two or more polypeptides of interest. In one example, the two or more nucleotide sequences are each linked to SG promoters. When two or more SG promoters are present in the sa-mRNA of the present disclosure, the promoters can be the same or different. For example, the two or more SG promoters are derived from the same alphavirus. In another example, the two or more SG promoters are derived from different alphaviruses.

When two or more SG promoters are present in the sa-mRNA of the present disclosure, the promoters can be the same or different. For example, the two or more SG promoters are derived from the same alphavirus. In another example, the two or more SG promoters are derived from different alphaviruses.

Internal ribosome entry site ( IRES )

As used herein, the term “internal ribosome entry site” or “IRES” refers to a sequence of nucleotides within a mRNA to which a ribosome or a component thereof, e.g., a 40S subunit of a ribosome, is capable of binding. An IRES need not necessarily comprise nucleic acid that induces translation of a mRNA (e.g., a start codon; AUG). An IRES suitable for use in the present disclosure will be apparent to the skilled person and/or are described herein and/or described in, for example, Martinez- Salas et al., 2018 Front Microbiol 8:2629.

In one example, the IRES is derived from encephalomyocarditis virus (EMCV). For example, the IRES is a wild-type IRES from EMCV.

In one example, the IRES is derived from a fibroblast growth factor 1A (FGF1A) IRES.

In one example, the IRES is derived from a fibroblast growth factor 2 (FGF2) IRES.

In one example, the IRES is derived from a platelet-derived growth factor (PDGF)

IRES. In one example, the IRES is derived from a vascular endothelial growth factor (VEGF) IRES.

In one example, the IRES is derived from an insulin-like growth factor 2 (IGF2) IRES.

In one example, the IRES is derived from a cricket paralysis virus (CrPV) IRES.

In one example, the IRES is derived from a hepatitis C virus (HCV) IRES.

In one example, the IRES is derived from a hepatitis A virus (HAV) IRES.

In one example, the IRES is derived from a poliovirus (PV) IRES).

In one example, the IRES is derived from a coxsackievirus B3 (CVB3) IRES.

In one example, the IRES is derived from an aichivirus (AiV) IRES. In addition, synthetic IRES elements have been described, which can be designed, according to methods know in the art to mimic the function of naturally occurring IRES elements (Chappell et al., 2000).

Self-cleaving peptide

As described herein, the proteins encoded by the open reading frames of the sa- mRNA are directly or indirectly linked to each other.

In some examples, the proteins are indirectly linked, e.g., via a linker.

In some examples, the linker is a 2A self-cleaving peptide.

As used herein, the term “2A self-cleaving peptide” refers to a peptide that allows for the translation of two linked proteins upstream and downstream of said peptide. The 2A self-cleaving peptide may allow read-through by the ribosome, resulting in translation of both the proteins as a fusion protein. Alternately, the ribosome may fall off after translation of the first protein, resulting in expression of the upstream protein alone.

Finally, the peptide may induce ribosomal skipping at the Glycine (G) and Proline (P) residues, whereby the ribosome skips the self-cleaving peptide and recommences translation at the start codon of the downstream protein. Thereby, expressing both the upstream and downstream protein as two separate proteins (Liu et al., 2017).

Non-limiting examples of 2A self-cleaving peptides suitable for the compositions and methods of the present disclosure include the peptide sequences from porcine teschovirus-1 2A (P2A), a foot-and-mouth disease virus (FMDV) 2A (F2A), an Equine Rhinitis A Virus (ERAV) 2A (E2A), a Thosea asigna virus 2A (T2A), a cytoplasmic polyhedrosis virus 2 A (BmCPV2A), a Flacherie Virus 2A (BmIFV2A), or a combination thereof.

Peptides or polypeptides The sa-mRNA of the present disclosure comprises an open reading that encodes a peptide or polypeptide (e.g., a pathogenic antigen).

In one example, the peptide or protein is a therapeutic protein for use in the treatment of cancer or tumor diseases, including cytokines, chemokines, suicide gene products, immunogenic proteins or peptides, apoptosis inducers, angiogenesis inhibitors, heat shock proteins, tumor antigens, beta-catenin inhibitors, activators of the STING pathway, checkpoint modulators, innate immune activators, antibodies, dominant negative receptors and decoy receptors, inhibitors of myeloid derived suppressor cells (MDSCs), IDO pathway inhibitors, and proteins or peptides that bind inhibitors of apoptosis;

In one example, the peptide r protein is a therapeutic protein for use in enzyme replacement therapy for the treatment of metabolic, endocrine or amino acid disorders or for use in replacing an absent, deficient or mutated protein, including Acid sphingomyelinase, Adipotide, Agalsidase-beta, Alglucosidase, alpha-galactosidase A, alpha-glucosidase, alpha-L-iduronidase, alpha-N-acetylglucosaminidase, Amphiregulin, Angiopoietins (Angl, Ang2, Ang3, Ang4, ANGPTL2, ANGPTL3, ANGPTL4, ANGPTL5, ANGPTL6, ANGPTL7), ATPase, Cu(2+)-transporting beta polypeptide (ATP7B), argininosuccinate synthetase (ASS1), Betacellulin, Beta-glucuronidase, Bone morphogenetic proteins BMPs (BMP1, BMP2, BMP3, BMP4, BMP5, BMP6, BMP7, BMP8a, BMP8b, BMP10, BMP15), CLN6 protein, Epidermal growth factor (EGF), Epigen, Epiregulin, Fibroblast Growth Factor (FGF, FGF-1, FGF-2, FGF-3, FGF-4, FGF-5, FGF-6, FGF-7, FGF-8, FGF-9, FGF-10, FGF-11, FGF-12, FGF-13, FGF-14, FGF-16, FGF-17, FGF-17, FGF-18, FGF-19, FGF-20, FGF-21, FGF-22, FGF-23), Fumarylacetoacetate Hydrolase (FAH), Galsulphase, Ghrelin, Glucocerebrosidase, GM- CSF, Heparin-binding EGF-like growth factor (HB-EGF), Hepatocyte growth factor HGF, Hepcidin, Human albumin, increased loss of albumin, Idursulphase (Iduronate-2- -acetylgalactosamine-4-sulfatase (rhASB; galsulfase, Arylsulfatase A (ARSA), Arylsulfatase B (ARSB)), N-acetylglucosamine-6-sulfatase, Nerve growth factor (NGF, Brain-Derived Neurotrophic Factor (BDNF), Neurotrophin-3 (NT-3), and Neurotrophin 4/5 (NT-4/5), Neuregulin (NRG1, NRG2, NRG3, NRG4), Neuropilin (NRP-1, NRP-2), Obestatin, phenylalanine hydroxylase (PAH), Phenylalanine ammonia lyase (PAE), Platelet Derived Growth factor (PDGF (PDFF-A, PDGF-B, PDGF-C, PDGF-D), TGF beta receptors (endoglin, TGF-beta 1 receptor, TGF-beta 2 receptor, TGF-beta 3 receptor), Thrombopoietin (THPO) (Megakaryocyte growth and development factor (MGDF)), Transforming Growth factor (TGF (TGF-a, TGF-beta (TGFbetal, TGFbeta2, and TGFbeta3))), VEGF (VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, VEGF-F und PIGF), Nesiritide, Trypsin, adrenocorticotrophic hormone (ACTH), Atrial- natriuretic peptide (ANP), Cholecystokinin, Gastrin, Leptin, Oxytocin, Somatostatin, Vasopressin (antidiuretic hormone), Calcitonin, Exenatide, Growth hormone (GH), somatotropin, Insulin, Insulin-like growth factor 1 IGF-1, Mecasermin rinfabate, IGF-1 analog, Mecasermin, IGF-1 analog, Pegvisomant, Pramlintide, Teriparatide (human parathyroid hormone residues 1-34), Becaplermin, Dibotermin-alpha (Bone morphogenetic protein 2), Histrelin acetate (gonadotropin releasing hormone; GnRH), Octreotide, hepatocyte nuclear factor 4 alpha (HNF4A), CCAAT/enhancer-binding protein alpha (CEBPA), fibroblast growth factor 21 (FGF21), extracellular matrix protease or human collagenase MMP1, Hepatocyte Growth Factor (HGF), TNF-related apoptosis-inducing ligand (TRAIL), opioid growth factor receptor-like 1 (OGFRL1), clostridial type II collagenase, Relaxin 1 (RLN1), Relaxin 2 (RLN2), Relaxin 3 (RLN3) and Palifermin (keratinocyte growth factor; KGF);

In one example, the therapeutic peptide or protein is a therapeutic protein for use in the treatment of blood disorders, diseases of the circulatory system, diseases of the respiratory system, cancer or tumor diseases, infectious diseases or immune deficiencies, including Alteplase (tissue plasminogen activator; tPA), Anistreplase, Antithrombin III (AT-III), Bivalirudin, Darbepoetin-alpha, Drotrecogin- alpha (activated protein C, Erythropoietin, Epoetin-alpha, erythropoietin, erthropoyetin, Factor IX, Factor Vila, Factor VIII, Lepirudin, Protein C concentrate, Reteplase (deletion mutein of tPA), Streptokinase, Tenecteplase, Urokinase, Angiostatin, Anti-CD22 immunotoxin, Denileukin diftitox, Immunocyanin, MPS (Metallopanstimulin), Aflibercept, Endostatin, Collagenase, Human deoxy-ribonuclease I, dornase, Hyaluronidase, Papain, L- Asparaginase, Peg-asparaginase, Rasburicase, Human chorionic gonadotropin (HCG), Human follicle-stimulating hormone (FSH), Lutropin-alpha, Prolactin, alpha- 1- Proteinase inhibitor, Lactase, Pancreatic enzymes (lipase, amylase, protease), Adenosine deaminase (pegademase bovine, PEG-ADA), Abatacept, Alefacept, Anakinra, Etanercept, Interleukin- 1 (IL-1) receptor antagonist, Anakinra, Thymulin, TNF-alpha antagonist,

In one example, the peptide or protein is a therapeutic protein selected from adjuvant or immuno stimulating proteins, including human adjuvant proteins, particularly pattern recognition receptors TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11; NODI, NOD2, NOD3, NOD4, NOD5, NALP1, NALP2, NALP3, NALP4, NALP5, NALP6, NALP6, NALP7, NALP7, NALP8, NALP9, NALP10, NALP11, NALP12, NALP13, NALP14,1 IPAF, NAIP, CIITA, RIG-I, MDA5 and LGP2, the signal transducers of TLR signaling including adaptor proteins including e.g. Trif and Cardif; components of the Small-GTPases signalling (RhoA, Ras, Rael, Cdc42, Rab etc.), components of the PIP signalling (PI3K, Src-Kinases, etc.), components of the MyD88-dependent signalling (MyD88, IRAKI, IRAK2, IRAK4, TIRAP, TRAF6 etc.), components of the MyD88-independent signalling (TICAM1, TICAM2, TRAF6, TBK1, IRF3, TAK1, IRAKI etc.); the activated kinases including e.g. Akt, MEKK1, MKK1, MKK3, MKK4, MKK6, MKK7, ERK1, ERK2, GSK3, PKC kinases, PKD kinases, GSK3 kinases, JNK, p38MAPK, TAK1, IKK, and TAK1; the activated transcription factors including e.g. NF-kB, c-Fos, c-Jun, c-Myc, CREB, AP-1, Elk-1, ATF2, IRF-3, IRF-7, heat shock proteins, such as HSP10, HSP60, HSP65, HSP70, HSP75 and HSP90, gp96, Fibrinogen, TypIII repeat extra domain A of fibronectin; or components of the complement system including Clq, MBL, Clr, Cis, C2b, Bb, D, MASP-1, MASP-2, C4b, C3b, C5a, C3a, C4a, C5b, C6, C7, C8, C9, CR1, CR2, CR3, CR4, ClqR, C1INH, C4bp, MCP, DAF, H, I, P and CD59, or induced target genes including e.g. Beta-Def ensin, cell surface proteins; or human adjuvant proteins including trif, flt-3 ligand, Gp96 or fibronectin, cytokines which induce or enhance an innate immune response, including IL-1 alpha, IL1 beta, IL-2, IL-6, IL-7, IL-8, IL-9, IL- 12, IL- 13, IL-15, IL-16, IL-17, IL-18, IL-21, IL-23, TNFalpha, IFNalpha (IFNa), IFNbeta (IFNb), IFNgamma, GM-CSF, G-CSF, M-CSF; chemokines including IL-8, IP- 10, MCP-1, MIP-1 alpha, RANTES, Eotaxin, CCL21; cytokines which are released from macrophages, including IL-1, IL-6, IL-8, IL- 12 and TNF-alpha; as well as IL-1R1 and IL-1 alpha; bacterial (adjuvant) proteins, including bacterial heat shock proteins or chaperons, including Hsp60, Hsp70, Hsp90, HsplOO; OmpA (Outer membrane protein) from gram-negative bacteria; OspA; bacterial porins, including OmpF; bacterial toxins, including pertussis toxin (PT) from Bordetella pertussis, pertussis adenylate cyclase toxin CyaA and CyaC from Bordetella pertussis, PT-9K/129G mutant from pertussis toxin, pertussis adenylate cyclase toxin CyaA and CyaC from Bordetella pertussis, tetanus toxin, cholera toxin (CT), cholera toxin B-subunit, CTK63 mutant from cholera toxin, CTE112K mutant from CT, Escherichia coli heat-labile enterotoxin (LT), B subunit from heat-labile enterotoxin (LTB) Escherichia coli heat-labile enterotoxin mutants with reduced toxicity, including LTK63, LTR72; phenol-soluble modulin; neutrophil-activating protein (HP-NAP) from Helicobacter pylori; Surfactant protein D; Outer surface protein A lipoprotein from Borrelia burgdorferi, Ag38 (38 kDa antigen) from Mycobacterium tuberculosis; proteins from bacterial fimbriae; Enterotoxin CT of Vibrio cholerae, Pilin from pili from gram negative bacteria, and Surfactant protein A and bacterial flagellins. In on example, the peptide or protein is a therapeutic protein used for reprogramming somatic cells into pluri- or omnipotent stem cells, including Oct-3/4, Sox gene family (Soxl, Sox2, Sox3, and Soxl5), Klf family (Klfl, Klf2, Klf4, and Klf5), Myc family (c-myc, L-myc, and N-myc), Nanog, and LIN28.

In one example, the peptide or polypeptide is an antigen from an infectious organism. For example, the peptide or polypeptide is an antigen which can induce an immune response in the subject.

In one example, the infectious organism is a virus. In one example, the virus causes a respiratory condition. For example, the virus is a coronavirus, respiratory syncytial virus (RSV), human metapneumo virus (hMPV) or parainfluenza virus (PIV).

Methods of expressing a peptide or polypeptide in a subject

The present disclosure provides a method of expressing a peptide or polypeptide in a subject, the method comprising administering the sa-mRNA, wherein the peptide or polypeptide encoded by the open reading frame(s) is linked to the open reading frame encoding the replicase or nsP4 with an IRES.

The T7 promoter which drives the expression of the replicase or nsP4 also drives expression of the downstream open reading frame encoding the peptide or polypeptide. Translation is initiated by the IRES encoded immediately 5’ to the open reading frame encoding the peptide or polypeptide. This allows for early expression of the peptide or polypeptide without the need for a subgenomic promoter (SGP).

Methods for determining the level of expression are known in the art and/or are described herein.

Methods of treating a disease or condition

The present disclosure provides methods of using the immunogenic composition or the pharmaceutical composition of the present disclosure as a vaccine.

The present disclosure also provides methods of treating or preventing a disease or condition in a subject comprising administering the sa-mRNA of the present disclosure. For example, the disease or condition is a respiratory virus infection, such as influenza, influenza virus infection, bronchiolitis, pneumonia, croup, a SARS-CoV-2 infection, COVID-19, ARDS, a RSV infection, a hMPV infection, a PIV3 infection and combinations thereof.

Methods of inducing an immune response in a subject The present disclosure provides a method of inducing an immune response in a subject, comprising administering the sa-mRNA of the present disclosure to a subject in need thereof. Upon administration of the sa-mRNA of the present disclosure to the subject, an immune response is induced that is specific to the peptide or polypeptide. The immune response may be sufficient to prevent subsequent infection in the subject, for example from coronavirus, RSV, hMPV or PIV. Additionally, the induction of the immune response may be sufficient to reduce or attenuate the severity of clinical symptoms, diseases or conditions associated with or resulting from future infection.

Following administration of the sa-mRNA of the present disclosure, the resulting immune response in the subject can be monitored by a number of conventional methods known in the art.

For example, the antibodies generated in the subject as a result of being administered the sa-mRNA of the present disclosure can be measured by obtaining biological samples from the subject, such as serum or plasma. Neutralizing antibody titres may then be measured using known methods in the art. Suitable methods include immunoasays, neutralization assays, flow cytometry-based assays and plaque reduction neutralization assays.

In one example, the sa-mRNA of the present disclosure, induces a humoral and/or a cell-mediated immune response.

SEQUENCES OF THE DISCLOSURE

The present disclosure includes the following non-limiting Examples. EXAMPLES

Example 1 - Materials & Methods

Generation of the self-amplifying RNA

DNA templates encoding self-amplifying RNAs described herein were produced in competent Escherichia coli cells transformed with a DNA plasmid. Individual bacterial colonies were isolated and the resultant plasmid DNA amplified in E. coli cultures. Following fermentation, the plasmid DNA was isolated using Maxiprep DNA kit and linearized by restriction digest. Restriction enzymes were removed using phenol/chloroform extraction and ethanol precipitation. mRNA was made by in vitro transcription from the linearized DNA template using a T7 RNA polymerase. Subsequently, the DNA template was removed by DNase digestion. Enzymatic capping was performed with CapO to provide functional mRNA. The resultant mRNA was then purified and resuspended in nuclease-free water.

Self- amplifying RNAs (Figure 1) were prepared using H5 from A/turkey /Turkey/ 1/2005. The following constructs were prepared:

• NSP1-4.SGP.H5 (F500.3)

• NSP1-4.IRES 6A.H5 (F903)

In vitro characterisation of the compositions

The self-amplifying RNAs were assessed for expression of the genes of interest that are expressed in the form of an antigen. Two-fold serial dilutions of self-amplifying mRNA constructs were electroporated into BHK-21, C2C12, HEK-293 cell lines. After about 17-19 hrs, cells were harvested and stained for antigen expression using suitable antibodies. The number of cells positive for antigen expression and the mean fluorescence intensities (MFIs) was measured by FACS. Data was analysed to calculate the specific potency values (the probability of successful transfection per unit of mass of RNA) and the MFI generated.

In vitro activity and potency of sa-mRNA was determined by FACs based on antigen co-expression.

Example 2 - Effect of linking gene of interest to nSP4 with IRES on sa-mRNA activity

A sa-mRNA construct was produced that contained no SGP and instead directly linked H5 to the nSP4 via an EMCV 6A IRES (F903). Figure 2A and Figure 2B show that F903 had increased H5 activity in BHK-21 cells when compared to the SGP- containing control (F500.3). Figure 3A and Figure 3B show that F903 had increased H5 activity in C2C12 cells when compared to the SGP-containing control (F500.3). Figure 4A and Figure 4B show that F903 had increased H5 activity in HEK-293 cells when compared to the SGP-containing control (F500.3). Figure 5 summarises the results of the trials represented in Figures 2-4.

Example 3 - Materials & Methods

Generation of the self-replicating RNA

DNA templates encoding self-replicating RNAs described herein were produced in competent Escherichia coli cells transformed with a DNA plasmid. Individual bacterial colonies were isolated and the resultant plasmid DNA amplified in E. coli cultures. Following fermentation, the plasmid DNA was isolated using Maxiprep DNA kit and linearized by restriction digest. Restriction enzymes were removed using phenol/chloroform extraction and ethanol precipitation. mRNA was made by in vitro transcription from the linearized DNA template using a T7 RNA polymerase. Subsequently, the DNA template was removed by DNase digestion. Enzymatic capping was performed with CapO to provide functional mRNA. The resultant mRNA was then purified and resuspended in nuclease-free water.

Self-replicating RNAs (Figure 6) were prepared using H5 from A/turkey /Turkey/ 1/2005 and NS1 from A/California/2009. The following constructs were prepared:

• NSPl-4.SGP.H5.SGPv2.Nl (F602)

• NSPl-4.SGP.H5.SGPv2.NSl (F693)

• NSPl-4.T2A.NSl.SGPv2.H5 (RD-01)

• NSP1-4.IRES 6A.NSl.SGPv2.H5 (RD-02)

• NSP1-4.IRES 6A.NSl.SGPv3.5.H5 (RD-03)

• NSP1-4.IRES 6A.NSl.SGPv4.5.H5 (RD-04)

• NSP1-4.IRES 6A.NSl.SGPv3.H5 (RD-05)

• NSP1-4.IRES 6A.NSl.SGPv4.H5 (RD-06)

• NSP1-4.IRES 6A.eGFP.SGPv2.H5 (RD-07)

• NSP1-4.IRES 6A.NSl.SGPv3.H5.SGPv2.Nl (RD-08)

• NSP1-4.IRES 6A.NSl.SGPv2.Firefly luciferase (RD-09)

• NSP1-4.IRES 6A.eGFP.SGPv2.CD45-F (RD- 10)

• NSP1-4.IRES 6A.eGFP.SGPv2.RSV-F DS Cavl (RD-11)

• NSPl-4.SGPv2.CD45-F (RD-12)

• NSP1-4.IRES 6A.NSl.SGPv2.CD90.1 (RD- 14)

• NSP1-4.IRES 6A.NSl.SGPv2.CD90.2 (RD- 15) NSPl-4.SGPv2.CD90.2 (RD- 16)

NSPl-4.SGPv2.CD90.1 (RD- 17)

In vitro characterisation of the compositions

The self-replicating RNAs were assessed for expression of the genes of interest that are expressed in the form of an antigen. Two-fold serial dilutions of self-amplifying mRNA constructs were electroporated into BHK-21, C2C12, HeLa or MRC-5 cell lines. After about 17-19 hrs, cells were harvested and stained for antigen expression using suitable antibodies. The number of cells positive for antigen expression and the mean fluorescence intensities (MFIs) was measured by FACS. Data was analysed to calculate the specific potency values (the probability of successful transfection per unit of mass of RNA) and the MFI generated.

Example 4 - Co-expression of Interferon antagonist from nSP4

Sa-mRNA constructs were produced that co-expressed the NS1 interferon antagonist with nSP4, while the H5 was expressed under the control of the SGP. As shown in Figure 6, in one construct separated the NS1 from nSP4 with a T2A selfcleaving peptide (RD-01) and in another the NS1 was separated from nSP4 with an EMCV 6A IRES (RD-02). Figure 7A shows a gel electrophoresis of these constructs, both as uncut and linearized vector. Figure 7B shows a gel electrophoresis of the products of IVT for both constructs. Figure 8A and Figure 8B show that RD-01 and RD-02 both had increased H5 activity in HeLa cells when compared to an H5 only control ((F500.3) and the control vector (F602). As shown in Figure 9A and Figure 9B, both RD-01 and RD-02 showed increased H5 activity in MRC5 cells when compared to an H5 only control ((F500.3). Figure 10A and Figure 10B show that RD-01 and RD-02 had no change in H5 activity in BHK21 cells due to immune-incompetency. As shown in Figure 11A and Figure 11B the expression of NS1 was highest for F693, followed by RD-02, with minimal expression observed for RD-01 in MRC5 cells.

Example 5 - Optimizing expression of gene of interest with subgenomic promoters

As shown in Figure 6 constructs were produced using the NS1 linked to nSP4 with an EMCV 6A IRES and H5 under the control of subgenomic promoters of various lengths. Figure 12A shows a gel electrophoresis of these constructs, both as uncut and linearized vector. Figure 12B shows a gel electrophoresis of the products of IVT for these constructs. Figure 13 and Figure 14 show sa-mRNA constructs with alternative genes of interest under the control of the subgenomic promoter. As shown in Figure 15A and Figure 15B, each of the constructs (RD-02, RD-04, RD-05, RD-06 and RD-07) showed increased H5 activity in C2C12 cells when compared to an H5 only control (F500.3), while RD-03 showed reduced H5 activity. Figure 16A and Figure 16B show that constructs RD-02, RD-03 and RD-04 all showed increased H5 activity in C2C12 cells when compared to an H5 only control (F500.3). Figure 17A and Figure 17B show that constructs RD-02 and RD-07 both showed increased H5 activity in C2C12 cells when compared to an H5 only control (F500.3), while RD-05 and RD-06 showed reduced H5 activity. As shown in Figure 18A and Figure 18B, each of RD-02, RD-03 and RD- 04 showed increased H5 activity in MRC5 cells when compared to an H5 only control (F500.3). Figure 19A and Figure 19B show that RD-02, RD-05 and RD-06 had increased H5 activity in MRC5 cells when compared to an H5 only control (F500.3), while RD-07 did not show increased H5 activity. As shown in Figure 20A and Figure 20B, each of RD-02, RD-03 and RD-04 showed increased H5 activity in MRC5 cells when compared to an H5 only control (F500.3). Figure 21A and Figure 21B show that each of RD-02, RD-05, RD-06 and RD-07 showed increased H5 activity in MRC5 cells when compared to an H5 only control (F500.3). Figure 22 summarises the results of the trials represented in Figures 15-21.

Claims

1. A self-amplifying RNA (sa-mRNA) comprising an open reading frame encoding a replicase or a non- structural protein 4 (nsP4) and an open reading frame encoding peptide or polypeptide, wherein the open reading frame encoding the peptide or polypeptide is co-expressed with the replicase or nsP4.

2. The sa-mRNA of claim 1, wherein the open reading frame encoding the peptide or polypeptide is linked with an internal ribosome entry site (IRES) that induces coexpression with the replicase or nsP4.

3. The sa-mRNA of claim 1, comprising a sequence encoding a protease cleavage site positioned between the peptide or polypeptide comprises and the replicase or the nsP4.

4. The sa-mRNA of claim 3, wherein protease cleavage site is a self-cleaving peptide.

5. The sa-mRNA of claim 2, wherein the IRES is selected from the group consisting of an EMCV IRES, a FGF1 IRES, a FGF2 IRES, a PDGF IRES, a VEGF IRES, an IGF2 IRES, a CrPV IRES, an HCV IRES, an HAV IRES, a PV IRES, a CVB3 IRES or an AiV IRES.

6. The sa-mRNA of claim 5, wherein the IRES is an EMCV 4A IRES, an EMCV 5A IRES, an EMCV 6A IRES, an EMCV 7A IRES, an EMCV 8A IRES, an EMCV 10A IRES or an EMCV 12A IRES.

7. The sa-mRNA of claim 6, wherein the IRES is an EMCV 6A IRES.

8. The sa-mRNA of claim 7, wherein the EMCV 6A IRES comprises a sequence set forth in SEQ ID NO: 1.

9. The sa-mRNA of claim 4, wherein the self-cleaving peptide is a 2A peptide.

10. The sa-mRNA of claim 4, wherein the self-cleaving peptide is a T2A peptide.

11. The sa-mRNA of claim 10, wherein the T2A peptide comprises a sequence set forth in SEQ ID NO: 5.

12. The sa-mRNA of claim 1 comprising in 5’ to 3’ order: an open reading frame encoding a replicase or a non- structural protein 4 (nsP4), an IRES, an open reading frame encoding a peptide or polypeptide, wherein the peptide or polypeptide is co-expressed with the replicase or nsP4.

13. The sa-mRNA of claim 1 comprising in 5’ to 3’ order: an open reading frame encoding a replicase or a non- structural protein 4 (nsP4), an EMCV IRES, an open reading frame encoding a peptide or polypeptide, wherein the peptide or polypeptide is co-expressed with the replicase or nsP4.

14. The sa-mRNA of claim 1, comprising in 5’ to 3’ order: an open reading frame encoding a replicase or a non- structural protein 4 (nsP4), a sequence encoding a selfcleaving peptide, an open reading frame encoding a peptide or polypeptide.

15. The sa-mRNA of claim 1, comprising in 5’ to 3’ order: an open reading frame encoding a replicase or a non-structural protein 4 (nsP4), a sequence encoding a T2A self-cleaving peptide, an open reading frame encoding a peptide or polypeptide.

16. The sa-mRNA of any claim 1 , wherein the peptide or polypeptide is/are antigen(s) from an infectious organism.

17. The sa-mRNA of claim 16, wherein the infectious organism is a virus.

18. The sa-mRNA of claim 17, wherein the virus causes a respiratory condition.

19. The sa-mRNA of claim 18, wherein the virus is a coronavirus or respiratory syncytial virus (RSV) or human metapneumo virus (hMPV) or parainfluenza virus (PIV).

20. The self- amplifying RNA of claim 19, wherein the antigen is selected from:

(i) Spike protein from a coronavirus or a prefusion stabilized version thereof or a receptor binding domain thereof;

(ii) F protein from RSV or a prefusion stabilized version thereof;

(iii) F protein from hMPV or a prefusion stabilized version thereof; (iv) F protein from PIV3 or a prefusion stabilized version thereof;

(v) Hemagglutinin from influenza; or

(vi) Neuraminidase from influenza.

21. The self-amplifying RNA of claim 19, wherein the RNA is multi-cistronic and comprises a sequence encoding hemagglutinin from influenza and a sequence encoding a neuraminidase from influenza.

22. The self-amplifying RNA of claim 19, additionally comprising one or more of the following:

(i) a sequence encoding spike protein from a coronavirus or a prefusion stabilized version thereof or a receptor binding domain thereof;

(ii) a sequence encoding F protein from RSV or a prefusion stabilized version thereof;

(iii) a sequence encoding F protein from hMPV or a prefusion stabilized version thereof; or

(iv) a sequence encoding F protein from PIV3 or a prefusion stabilized version thereof.

23. A method of expressing a peptide or polypeptide in a subject, the method comprising administering the sa-mRNA of claim 1, wherein the peptide or polypeptide is encoded by the open reading frame(s) linked to the replicase or nsP4.

24. A method of treating a disease or condition, the method comprising administering the sa-mRNA of claim 1, wherein a therapeutic peptide or polypeptide is encoded by the open reading frame(s) linked to the replicase or nsP4 and expression of the therapeutic peptide or polypeptide treats the disease or condition.

25. A method of inducing an immune response in a subject, the method comprising administering the sa-mRNA of claim 1, wherein an antigen is encoded by the open reading frame(s) linked to the to the replicase or nsP4 and expression of the antigen induces the immune response.

26. Use of the sa-mRNA of claim 1 in the manufacture of a medicament for expressing a peptide or polypeptide in a subject.

27. Use of the sa-mRNA of claim 1 in the manufacture of a medicament for treating a disease or condition in a subject.

28. Use of the sa-mRNA of claim 1 in the manufacture of a medicament for inducing an immune response in a subject.

29. The sa-mRNA of claim 1 for use in expressing a peptide or polypeptide in a subject.

30. The sa-mRNA of claim 1 for use in treating a disease or condition in a subject.

31. The sa-mRNA of claim 1 for use in inducing an immune response in a subject.